Genetic characterization of <Emphasis Type="Italic">Pepino mosaic virus</Emphasis> isolates from Belgian greenhouse tomatoes reveals genetic recombination

Over a period of a few years, Pepino mosaic virus (PepMV) has become one of the most important viral diseases in tomato production worldwide. Infection by PepMV can cause a broad range of symptoms on tomato plants, often leading to significant financial losses. At present, five PepMV genotypes (EU, LP, CH2, US1 and US2) have been described, three of which (EU, LP and US2) have been reported in Europe. Thus far, no correlation has been found between different PepMV genotypes and the symptoms expressed in infected plants. In this paper, the genetic diversity of the PepMV population in Belgian greenhouses is studied and related to symptom development in tomato crops. A novel assay based on restriction fragment length polymorphism (RFLP) was developed to discriminate between the different PepMV genotypes. Both RFLP and sequence analysis revealed the occurrence of two genotypes, the EU genotype and the CH2 genotype, within tomato production in Belgium. Whereas no differences were observed in symptom expression between plants infected by one of the two genotypes, co-infection with both genotypes resulted in more severe PepMV symptoms. Furthermore, our study revealed that PepMV recombinants frequently occur in mixed infections under natural conditions. This may possibly result in the generation of viral variants with increased aggressiveness.     

Simultaneous detection and identification of <Emphasis Type="Italic">Pepino mosaic virus</Emphasis> (PepMV) isolates by multiplex one-step RT-PCR

A RT-PCR was developed for the simultaneous detection and identification of three groups of Pepino mosaic virus (PepMV): European/Peruvian, Chilean 1/US1 and Chilean 2/US2 groups, followed by a restriction analysis that allowed the separation of the European, Peruvian, Chilean 2 and US2 isolates (patent pending). The multiplex RT-PCR reaction was performed by a mix of six primers that amplified a part of the RNA-dependent RNA polymerase gene of PepMV plus an internal control. Amplifications resulted in a 980 bp, 703 bp or 549 bp PCR product for European/Peruvian, Chilean 1/US1 or Chilean 2/US2 groups, respectively. For the identification of the isolates present within the European/Peruvian and Chilean 2/US2 groups, the amplified PCR fragments were directly digested with SacI enzyme. The multiplex RT-PCR method presented higher sensitivity to detect CH1/US1 isolates in field samples than the RFLP-PCR method described by Hanssen et al. (European Journal of Plant Pathology 121:131–146, 2008). The detection limit observed with the multiplex RT-PCR was equal to or 3,125 times higher when compared to single RT-PCR or ELISA-DAS and molecular hybridisation methods, respectively. The use of the multiplex RT-PCR method in routine analysis of field tomato samples allowed the detection of 36.2 and 33.4% more positives when compared to the serological and molecular hybridisation methods, respectively, and the identification of plants infected with one, two or three isolates of PepMV.     

Pepino mosaic virus isolates and differential symptomatology in tomato

Based on a survey conducted in commercial tomato production in Belgium in 2006, four Pepino mosaic virus (PepMV) isolates that differed in symptom expression in the crop of origin were selected for greenhouse trials. The selected isolates were inoculated onto tomato plants grown in four separate plastic tunnels. PepMV symptom development was assessed regularly and extensive sampling followed by ELISA analyses, genotyping and sequencing was performed to study viral presence and variation in PepMV sequences throughout the trial period. Two isolates (EU-mild and CH2-mild) that were selected based on mild symptom expression in the crop of origin caused only mild symptoms in the trial, while two other isolates (CH2-aggressive and EU + CH2) that were selected for severe symptom display, caused considerably more severe symptoms. Sequence homology between CH2-mild and CH2-aggressive was as high as 99·4%. Results of this study show that differential symptom expression can, at least partially, be attributed to the PepMV isolate, which may be related to minor differences at the nucleotide level between isolates.     

Genetic variation and evolutionary analysis of Pepino mosaic virus in Sicily: insights into the dispersion and epidemiology

Pepino mosaic virus (PepMV) is a highly infectious potexvirus that causes a severe disease in tomato (Solanum lycopersicum) crops worldwide. In Sicily, the first outbreak was detected in a single greenhouse in 2005 and it was promptly eradicated. However, in 2008, a large number of greenhouses were simultaneously affected, and it was impossible to eradicate or control the virus. This study addressed the dispersion and the genetic diversity of PepMV isolates obtained from the outbreak in Sicily, in comparison with worldwide PepMV isolates, to gain insight into the factors determining the evolution and epidemiology of the virus. A total of 1800 samples from plants with and without symptoms were collected in the Sicilian provinces of Agrigento, Caltanissetta, Palermo, Ragusa, Siracusa and Trapani. Three isolates collected at different times were biologically characterized. The incidence of the virus increased rapidly from 13% in 2011 to 63% in 2013, and phylogenetic analysis showed that all Sicilian isolates of PepMV belonged to the CH2 strain, one of the six strains previously described. Nucleotide diversity of the Sicilian isolates was low, thus suggesting rapid spread and genetic stability.